Synthetic Silica Nano‐Organelles for Regulation of Cascade Reactions in Multi‐Compartmentalized Systems

Abstract In eukaryotic cells, enzymes are compartmentalized into specific organelles so that different reactions and processes can be performed efficiently and with a high degree of control. In this work, we show that these features can be artificially emulated in robust synthetic organelles constructed using an enzyme co‐compartmentalization strategy. We describe an in situ encapsulation approach that allows enzymes to be loaded into silica nanoreactors in well‐defined compositions. The nanoreactors can be combined into integrated systems to produce a desired reaction outcome. We used the selective enzyme co‐compartmentalization and nanoreactor integration to regulate competitive cascade reactions and to modulate the kinetics of sequential reactions involving multiple nanoreactors. Furthermore, we show that the nanoreactors can be efficiently loaded into giant polymer vesicles, resulting in multi‐compartmentalized microreactors.


Synthesis of enzyme-loaded silica nanocapsules as organelle-mimetic modules
Silica nanocapsules were synthesized by using an inverse (water-in-oil) miniemulsion technique (see Figure S1). Enzymes (e.g. GOx or HRP) were directly encapsulated in the aqueous core of the nanocapsules during capsule formation.
Specifically, an aqueous phase and an oil phase were prepared separately. The enzyme was pre-dissolved at a concentration of 5 mg mL -1 in the aqueous phase (sodium phosphate buffer, 10 mM, pH 7.4). The oil phase was composed of a solution of 26.7 mg mL -1 PGPR in cyclohexane. For preparing emulsions, 30 mL oil solution was poured into the vial containing 1 mL aqueous phase under magnetic stirring at 1000 rpm. The oil-water mixture was homogenized with a T25 Ultra-Turrax at 13,000 rpm for 1 min. The emulsion was then processed in a microfluidizer (LM10, Microfluidics Corporation) cooled with water. The microfluidizer is equipped with an air-driven intensifier pump that supplies the desired pressure to the emulsion stream. The processed emulsions were collected from the outlet flow and were fed again to the inlet reservoir. Each emulsion was processed for two cycles of microfluidization. After the emulsification, a mixture of 335 μL of TMOS and 100 μL of APTES were added to the emulsion in 10 seconds under stirring at 1000 rpm. Samples were kept stirring at room temperature for another 12 h before the sol-gel reactions were completely finished.
The synthesized nanocapsules in cyclohexane phase were then transferred to an aqueous medium. For this purpose, 6 mL of Lutensol AT50 solution (0.3 wt% in water) was prepared and placed in a sonication bath. Next, 0.6 mL of nanocapsule-cyclohexane dispersion was added dropwise to the Lutensol solution in 5 min under shaking. The mixture was kept stirring in an open flask (without lid) at 1000 rpm and room temperature for 24 h to evaporate the cyclohexane. The samples in aqueous medium were then washed 3 times with water by centrifuging at 1660 g for 20 min at 10 °C. Supernatants from the centrifugation step were discarded and fresh water was added. The dispersions were then homogenized by repetitive pipetting.

Characterization of silica nanocapsules
The average size and size distribution of nanocapsules were measured by dynamic light scattering (DLS) at 25 °C on a Nicomp 380 submicron particle sizer (Nicomp Particle Sizing Systems, USA) at a fixed scattering angle of 90°. Zeta potential measurements were performed in 10 −3 M potassium chloride solution at pH 6.8 and 25 °C with a Malvern Zeta sizer (Malvern Instruments, UK). Solid content of the capsule dispersion was measured gravimetrically. The morphology of nanocapsules was examined with a Jeol 1400 (Jeol Ltd, Tokyo, Japan) transmission electron microscope (TEM) operating at an accelerating voltage of 120 kV.
Samples for TEM were prepared by casting the diluted dispersions (solid content: ~0.01 wt%) on carbon layer-coated copper grids.

Encapsulation efficiency of enzymes
Encapsulation efficiency of enzymes in the nanocapsules was determined by separating the capsules from non-encapsulated enzymes in the dispersion by centrifugation. Firstly, 1 mL of nanocapsule dispersion (solid content = 1.0 wt%) was centrifuged at 1760 g for 40 min at 10 °C. The pellet was collected, and the supernatant (SN) was transferred to a new tube for another centrifugation step. The centrifugation was repeated for three times. The enzyme contents in the three pellets and in the final supernatant were determined by using BCA (bicinchoninic acid) protein assay ( Figure S4). 5 μL of the sample was incubated with 150 μL of BCA working reagent at 37 °C for 30 min. The absorbance at 562 nm was recorded by plate reader (TECAN M1000). Because of the silica absorption at this wavelength, the final enzyme content was determined by subtracting the total absorption with the absorption of empty silica NRs as the control sample. The encapsulation efficiency was calculated by using the following equation: where Pn represents the pellet collected at centrifugation step (n), and SNn represents the supernatant obtained from the centrifugation step (n).

Fluorescent labeling of enzymes
Firstly, a stock solution of BDP FL-NHS ester or Cy5-NHS ester in DMSO was prepared.
Enzymes (GOx or HRP) were dissolved in 0.1 M sodium bicarbonate solution at a concentration of 10 mg/mL. The dye solution was mixed with the enzyme solution at the volume ratio of 1:9. The final molar ratio of dye to the enzymes is 8:1. The mixture was stirred at 1000 rpm for 3 min and then incubated at 25 o C for 4 h. Afterwards, the samples were purified by using a gel-filtration column (Zeba™ Spin Desalting Columns, 7K MWCO, 10 mL; Thermo Fisher 89893). Before use, the column was cleaned three times with buffer by centrifugation at 1000g for 2 min. The labelled samples were then placed in the column and centrifugated at 1000g for 2 min. The purified samples were redispersed in buffer solution and collected in a new vial for further use of preparing nanocapsules.

Nanoreactor permeability test
The passive diffusion of small water soluble molecules was demonstrated by fluorescence intensity measurements performed on a TECAN M1000 plate reader. A reaction cocktail was prepared with following composition: 940 µl PBS buffer + 20 µl Glucose + 3.3 Amplex Red. After homogenization, 100 µl of the reaction cocktail was placed in separate wells. Then 2.5 µl of nanocapsule dispersion was added to each well to start the reaction. The following experimental set up was used: 555 nm excitation, 595 nm emission. A kinetic cycle was performed for 1 hour with measurements taken every 15 seconds. The results are shown in Figure S7.

Solution
Concentration PBS buffer 10 mM Glucose in deionized water 10 mM Amplex Red in DMSO 1 mM HRP and GOx Nanocapsules in water ca. 0.24 g/L* * solid content of stock dispersion: 0.97 wt%

Batch-to-batch reproducibility
A reaction cocktail was prepared as a stock solution in the following composition: 940 µl PBS buffer + 20 µl Glucose + 3.3 Amplex Red. After homogenization, 100 µl of the reaction cocktail was placed in each well. Then either 2.5 µl of HRP and GOx nanocapsules or 1.25 µl HRP nanocapsules + 1.25 GOx nanocapsules were added to each well to start the reaction. The measurements were performed using new and distinct batches of nanocapsules. The following experimental set up was used: 555 nm excitation, 595 nm emission. A kinetic cycle was performed for 1 hour with measurements taken every 15 seconds. The results are shown in Figure S8.

Photolithography
Photolithography is the process of transferring geometric shapes on a mask to the surface of a silicon wafer. The steps involved in the photolithographic process were wafer cleaning; barrier layer formation; photoresist application; soft baking; mask alignment; exposure and development; and hard baking. In the first step, the wafers were chemically cleaned to remove particulate matter on the surface as well as any traces of organic, ionic, and metallic impurities.
Then the wafers were pre-backed on a hotplate at 200 °C for 5 minutes. Then, the wafers were spin-coated with photoresist SU-83050 at 2200 rpm for 30 seconds to achieve a height of approximately 70 μm and with photoresist SU-83030 at 1800 rpm for 30 seconds again to achieve a height of 40 μm. Then the wafers were pre-baked on a hotplate for 1 minute at 65 °C, and soft-baked for 20 minutes at 95 °C and in the end for cooling down baked again at 65 °C for 1 minute. Soft baking is the step during which almost all of the solvents are removed from the photoresist coating. The wafer and the photoresist were then aligned with a film mask containing the chip designs. Once the mask had been accurately aligned with the pattern on the wafer's surface, the photoresist is exposed through the pattern on the mask with a high intensity ultraviolet light for 5 seconds. The wafer and the UV-treated photoresist were post-baked on a hotplate for 1 minute at 65 °C, then baked at 95 °C and again for 1 minute at 65 °C. Hardbaking is the final step in the photolithographic process. This step is necessary in order to harden the photoresist and improve adhesion of the photoresist to the wafer surface. The unexposed photoresist was dissolved by soaking the wafer in a developer for 10 minutes to 15 minutes. The finished wafer was washed with isopropanol and dried with nitrogen. The wafer was inspected under a microscope and the height was measure on the 4 poles of the wafer.

PDMS chip production
For the production of microfluidic chips, the silicon wafers with the channel molds were placed in a glass dish and covered with from 5-10 mm high mixture of liquid PDMS and curing agent in a 9:1 ration (for 1 glass dish around 40 mg of mixture was needed). The glass dish with the wafer and the liquid mixed PDMS and curing agent solution was degassed via a conventional vacuum pump and chamber until all air bubbles disappeared. The wafers were then placed at 80 °C for at least 2 hours in order to improve molecular cross-linking. Once hardened, individual PDMS-replicas were cut out using a scalpel. The three inlet channels and the outer channel are pierced with a syringe needle to create a connection between the channels and the later outside. The side of the replica with the channels was then covered with tape until the activation in the plasma cleaner to protect in from dust particles and to remove dirt from the surface. A large cover glass slide is thoroughly cleaned with 70 % ethanol. Then the glass slide is activated together with the PDMS-replica (channels from the upper side in the device) in a plasma cleaner (Diener Electronic Plasma-Surface-Technology, Model Femto) for exactly 30 seconds at the 30% power which equals to 30 W. The activated glass slide was therefore pressed carefully on the activated side of the PDMS-replica with the microfluidic channels and again baked at 80 °C for at least 90 minutes (excluding the baking time after coating) to improve the creation of the covalent bond. Finished chips were carefully stored in a glass dish protected from dust particles and dirt. Before usage the chips were inspected under microscope for irregularities or dirt inside the channels.

Chip coating
The chip coating is required in double emulsion chips to enable a stable emulsion at the second junction and prevent the middle fluid from sticking to the otherwise hydrophobic walls of the outlet channel. This coating method required the chips to be coated straight after activation. After production, the coating of the outer channel is inspected on a microscope.

Fluid compositions
Inner and outer solutions were prepared fresh from stock solutions for all experiments and not used longer than one week. The respective stock solutions were not older than one week either and were prepared in ddH2O. Sucrose, fructose and glucose were prepared at a concentration of 300 mM and their pH level was measured and maintained before every experiment in the range of 7-9. 1 M NaOH solution was used for correction of the pH value of the stock solutions.
The middle solution consisted of 10 mg/mL of PB22-PEO14 in oleyl alcohol as stock solution.
1 mM Amplex red in DMSO was prepared as a stock solution for initiation of the resorufin production reaction. For microscopy, the vesicle dispersion was transferred to an aqueous fructose solution (300 mM).

Microscopy
Confocal laser scanning microscopy (CLSM) images and videos were acquired using a Leica  Figure S1. Schematic illustration of the preparation of silica nanoreactors with in-situ loaded enzymes by using an inverse (water-in-oil) miniemulsion polymerization process.     Figure S7. Fluorescence intensity measurements of resorufin production by (GOx+HRP)@NRs without removal of the NRs (non-filtered, NF) and after removal of the NRs (filtered, F). Comparison between NF and F proves that the product can diffuse out of the NRs. Samples were measured using two distinct batches of nanoreactors. The signals from F1 and F2 in the plot correspond to the amount of resorufin produced up to the moment when the NRs were removed. Since no reaction could proceed without the NRs, the signals remained constant. Additionally, the signals were significantly higher than the background signal, indicating that the product (resorufin) must have left the interior of the porous NRs. In the NF samples, the reaction proceeded as expected, eventually reaching a plateau after the substrates were consumed.